Picornaviruses as well as other positive strand RNA viruses utilize RNA templates, viral and host proteins, and the architecture provided by cytoplasmic membranes of the infected cell to orchestrate a complex series of macromolecular events that lead to the production of progeny viral RNAs. Poliovirus, coxsackievirus, and human rhinovirus are members of the Picornaviridae that share a common RNA replication strategy during their intracellular replication cycles. This proposal outlines new experimental approaches to dissect the mechanisms that underlie this complicated process. These approaches will focus on the assembly and dynamics/maturation of viral RNA replication complexes during the course of infection. The proposed experiments build on the knowledge that viral protein 3CD is an integral component of picornavirus RNA replication complexes as are host cell proteins hnRNP C and PCBP. Dual tagged forms of poliovirus 3CD will be used to isolate viral RNA replication complexes at different stages of infection and under altered conditions of proteolytic processing. Complexes will be analyzed for viral and host proteins known to play roles in picornavirus RNA replication. In addition, an unbiased, high-resolution proteomics approach will be employed by performing mass spectrometry on such isolated complexes. The overall approach will be comprehensive in its analyses because the functional significance of newly-identified protein players will be tested both by infections of human cell lines and by cell-free RNA replication reactions that can lead to the synthesis of infectious poliovirus or human rhinovirus. The first aim of this proposal is to determine how binding of hnRNP C to both termini of poliovirus negative-strand RNA intermediates facilitates template recognition and assembly of RNA replication complexes. The second aim of the proposal will define the complete proteome of specific poliovirus RNA replication complexes using dual-affinity tagged polypeptide 3CD and mass spectrometry. The experiments proposed in this aim will also determine the RNA replication complex proteome for human rhinovirus, which should reveal unifying principles for which viral and host proteins comprise the core picornavirus RNA replication complex and those that are unique for human rhinovirus or poliovirus. In addition to providing new mechanistic insights for how cytoplasmic RNA viruses replicate their genomic RNAs, the proposed studies should reveal novel protein-protein and protein-RNA interfaces that will serve as important targets for the development of anti-viral therapeutic approaches.